710
K. J. PALMER, R. C. MERRILL, H. S. OWENS, AND M. BALLANTYNE
the difficulty of drying acetone (2) and Lsnnung’s method of drying (distillation from potassium carbonate and protection by calcium chloride), it would be surprising if a much higher degree of dryness n-ere obtained. Lannung’s own criterion of dryness-conductivity of saturated sodium chloride solution-suggests a variation of 0.05 per cent moisture in the acetone used. If this view is correct, the conductivity of saturated solutions of a salt such as cesium fluoride would be a simple and sensitive method of determining traces of moisture in acetone and their conductivity in anhydrous acetone would be of a smaller order of magnitude than reported by Lannung. REFERESCES
(1) LANNUNG, A.: Z. physik. Chem. 161, 255, 269 (1932). (2) TIMMERMANS, J., A N D GILLO,L . : Roczniki Chem. 18, 812 (1938).
AN X-RAY DIFFRACTIO?:
INVESTIGATIOS OF PECTINIC AND PECTIC ACIDS
K. J. PALMER, R. C. MERRILL, H. S. OWENS, AND M. BALLANTYNE Weelern Regional Research Laboratory,’ Albany, Californaa Received January 8, 1947
It has been recognized for some time (10) that pectinic acid is composed of an essentially linear polygalacturonide chain of length sufficient for the production of fibers. However, the only published x-ray data on fibers of this important natural high polymer are those of Wuhrmann and Pilnik (19). In 1933 Van Iterson and Corbeau (18) obtained optically negative, uniaxial. birefringent fibers by spinning concentrated aqueous solutions of commercia. citrus pectin into an alcohol-ether mixture. These authors claimed that x-ray photographs of these fibers showed the presence of oriented crystallites. Henglein and Schneider (7) have since reported that x-ray photographs of nitropectin fibers show weak crystallite orientation. Kringstad and Lunde (9) have published a power photograph of a pectinic acid, but they gave neither the values of the spacings nor data from which they could be calculated. Astbury and Bell (2) have reported results obtained by K. L. Scott on commercial lemon pectin. Only Wuhrmann and Pilnik (19) have attempted to deduce any structural information from their x-ray photographs. These authors suggest, on the basis of the x-ray patterns obtained from oriented films and fibers of pectinic and pectic acids, that the fiber identity period is about 8.8 A. In the present paper it will be shown that this value is too small, the fiber identity period actually being about 13 A. This value of 13 A. is similar to that found to occur in sodium pectate (14, 15). tion
Bureau of Agricultural and Industrial Chemistry, Agricultural Research AdministrsU. S. Department of Agriculture.
X-RAY DIFFRACTIOX STUDY O F I’ECTINIC
AKD PECTIC ACIDS
711
-
The present paper is concerned with an analysis and interpretation of data obtained from x-ray patterns of pectinic acids (from 11 to 1 per cent methoxyl content) and pectic acid ( < 1 per cent methoxyl content), in the form of both powders and fibers. Although a method has been developed which produces fibers having a rather high degree of molecular orientation, the quantity of diffraction data obtained is unfortunately still meager. This has been partially compensated for by studying the variation of the x-ray patterns with moisture and methoxyl content. In spite of the lack of general information with regard to the chemical structure of pectin, the x-ray data obtained allow some interesting conclusions to be drawn regarding the configuration of the polygalacturonide chain. EXPERIMESTAL
The commercial citrus and applepectinswere deashed by means of ion-exchange resins as described in the literature (12). The rest of the samples were kindly supplied by Dr. T. H. Schultz and H. Lotzkar and were prepared as follows: Samples L51 and L65 were extracted from lemon peel at p H 3.5 with three volumes of water containing 12 g. of “sodium hexametaphosphate” (Calgon) per kilogram of peel for 20 min. a t 90-95°C. The filtered extract was precipitated in ethanol a t pH 1.0 to 1.5. The precipitate was washed wit,h 55 per cent ethanol until free of chloride, then hardened with 95 per cent ethanol, and dried in U ~ C U Oa t 60°C. The ether samples were deesterified by citrus pectinesterase acting in situ in lemon peel,’ as described previously (13). Deashing was accomplished in the same way as for samples L51 and LG5. Samples L43 and L69 were extracted by the procedure of Baier and Wilson (3). Sample L43C was precipitated in neutral alcohol and received no deashing treatment. The analytical and viscosity data given in table 1 were obtained by methods previously described (12). The per cent water in the equilibrated samples was determined by measuring the loss in weight of samples dried in vacuo at 70°C. for 16 hr. X-ray photographs of the powders xere taken after the samples had been equilibrated with an atmosphere having a relative humidity of 49 per cent by means of a vacuum desiccator containing the proper concentration of sulfuric acid. Column 6 of table 1gives the per cent water sorbed by these samples when in equilibrium with an atmosphere having a relative humidity of 49 per cent. Column 2 of table 1 gives the methoxyl content and column 3 the ash content of the samples. The per cent uronide is given in column 4 for those samples for which this data is known, XT-hile column 5 lists the values of the intrinsic viscosity. The fibers were prepared by using 2-4 per cent solutions which had been freed from bubbles and suspended solids by centrifugation. The resulting solution was forced through a 1-mm. round orifice into a coagulating bath composed of an equal-volume mixture of alcohol and ether. The fibers mere usually partially dehydrated for 5-15 min. in an alcohol bath and then suspended in air to dry. During drying they were stretched by adding small weights. The dry fibers were then given an additional stretch, varying between 40 and 100 per cent, by suspending them with weights attached in the vapor of a boiling alcoholwater mixture. The ratio of alcohol to water was found t o be very critical
712
K. J. PALMER, R . c . MERRILL, H. s. OWENS, AND M. BALLANTYNE
for a given fiber with a given weight attached. In general, the latitude of the alcohol concentration between no stretch and elongation until break was only about 2-3 per cent. The per cent elongation could be controlled by using intermediate concentrations. In our particular arrangement, using six fibers about 0.5 mm. in diameter and with a 100-g. weight attached, the optimum concentration of alcohol in the liquid was found to lie between 4 and 7 per cent. Ana S411PI.E
L51. . . . . . . . . . . . Commercial citrus pecti (deaahed) . . . . . . . . . . . . L65 . . . . . . . . . . . . . . . . . . . L25. . . . . . . . . . . . . . . . . . . . L25. . . . . . . . . . . . . . . . . . . . Commercial apple pecti (deashed) . . . . . . . . . . . .
ical data
01
TABLE 1 ieclinic and pectic acid samples
CHtO
ASH
URONIC ACID ANRPDRIBE
per cenf
pn C f n l
per crnt
10.9
0.4
10.7 10.5 10.7 10.7
0.2 1.o 0.6 0.6
VATEP CONTXNT
AT R.H. 49 PEP CEHT
per cfnt
9.3 85 80 80 80
4.2
7.9 8.7 8.7
10.9
0.2
84
6.4
L64. . . . . . . . . . . . . . . . . . . .
7.4
0.4
79f
5.5
L37P. . . . . . . . . . . . . . . . . . .
5.8
0.4
Sot.
5.8
L81. . . . . . . . . . . . . . . . . . . .
4.5
0.5
got
5.2
L67. . . . . . . . . . . . . . . . . .
2.7
0.3
81t
3.5
L66. . . . . . . . . . . . . . . . . . . .
L43 . . . . . . . . . . . . . . . . . L43c.. . . . . . . . . . . . . . . . . .
1.4 0.6 0.6
0.7 0.4 15.0
82 t 82
2.2 4.2
L69. . . . . . . . . . . . . . . . . . . .
0.7
0.2
82
3.7
17.4: 15.8t 18.2" 16.0t 15.8t 16.5* 18.0* 18.6* 16.6t
17.7* 16.07
18.2* 16.3t 18.4* 15.8t 16.0t 16.6t 23.4* 18.3* 15.5t
* Desorption.
t Sorption.
1Estimated.
The density was determined by suspension in a toluene-ethylene bromide mixture. The values found are listed in the bottom row of table 2 . The density found by this method is in good agreement with that found for powdered pectin by the helium gas displacement method (17). The value 1.18 for the specific gravity of orange pectin a t 20°C. (11) is either an apparent bulk specific gravity or too low because of incomplete penetration of sample by the pycnometer liquid.
713
X-RAY DIFFRACTION STUDY O F PECTINIC A S D PECTIC ACIDS
X-ray photographs of the fibers were made in the usual way with CuK. radiation filtered through thin nickel foil. The powders, after being brought to equilibrium with an atmcsphere having a relative humidity of 49 per cent, were quickly placed in methyl methacrylate capillaries. The capillaries were sealed to a dose-fitting copper wire a t one end and sealed cff a t the other end by means of melted beeswax. The methyl methacrylate capillaries were made by the method of Fricke and coworkers ( 5 ) . RESULTS
The x-ray patterns of powders of the various pectinic and pectic acids are similar in general appearance (figure 1). Lon-er methyl ester content, hon-ever, usually gives rise to somewhat sharper rings, as would be expected because TABLE 2 X - r a y spacings a n d d e n s i t y of pectinic a n d pectic acids in e q u i l i b r i u m a t 49 per cent relative h u m i d i t u
I [ I I X-ray spac-
7.0t
L43
U3C
-
L69
--
-
1.617.11
.58
4.37 ing in A , . . 1 43.0: . 1 8 i . 2 2 4.18 4.18 4 . 2 3.06 3.04 3.09 3.0!
l:ll
..491.54 1.51 1.50
I
,144.19 .05 2.98
.12 .98
1.52 -
* Measurement made at sharp inner edge of reflection.
t Measurement made at center of reflection.
the more uniform chains can pack together in a more regular manner. In general, only three distinct rings have been observed from deashed high-molecular-weight pectinic and pectic acids, although under proper conditions one or more additional rings appear. For example, when a high-molecular-weight pectic acid, 15 hich gives the usual x-ray pattern showing three rings, is heated for several hours a t SO'C., the resulting heat-degraded pectin, after careful washing with water t o remove any lorn-molecular-weight products, gives an x-ray pattern which exhibits as many as fifteen sharp rings. These heatdegraded products are now under investigation, and the results will be published in the near future. The three distinct characteristic rings consist of an intense, rather diffuse inner ring, a moderately intense, rather sharp intermediate ring, and a weak outer ring (figure 1). The spacings calculated for these rings for the samples listed in table 1 are given in table 2.
715
X-RAY DIFFRACTIOS STUDY O F PECTISJC AXD PECTIC ACIDS
relative humidity of 49 per cent and had the vater contents listed in the last column of table 1. A plot of the interplanar spacing obtained from the intense inner ring versus methoxyl content a t a constant nater content of 16 per cent is shonn in figure 2. This latter spacing is also a function of the nater content. The effect of moisture, however, will be considered in more detail in another place (16). One other point of interest is illustrated by the t n o samples L25 and commercial citrus pectin. When these samples are humidified and then placed in an atmosphere having a humidity of 49 per cent until their neight becomes constant, both exhibit inner rings n hich correspond to spacings of about 10.9 A. (table 2). When these samples are dried in a i0"C. vacuum oven and then brought to equilibrium a t 49 per cent relative humidity, the inner 7.00 '5
a
I
6.90
-
!-I 0 6.80 w c
-
Y
zS#
I
I
I
I
I L51
-
-
J Z
2
0
W
6.50
-
I 2
I
I
I
I
I
4
6
8
IO
12
METHOXYL CONTENT
(%I
FIG.2. Plot of intense equatorially accentuated x-ray reflection against methoxyl content for samples having a moisture content of 16 per cent.
ring is no longer observed. This effect is reproducible on subsequent cycles. The 10.9 spacing probably results from the higher degree of crystallinity made possible because the hydrated chains are free to move Jvith respect to one another and therefore assume a more regular arrangement. The fiber diagram of pectic acid L69, which is typical of those obtained from the other samples, is reproduced in figure 3 and data are given in table 3. I t is evident from this photograph that the 6.5-6 9 A. ring becomes equatorially accentuated, n hile the two rings a t approximately 4 A. and 3 A. become meridionally accentuated. I n addition, the most highly oriented fibers exhibit an additional meridionally accentuated arc giving a spacing of about G.1 A. and a very diffuse equatorial spot. This latter spot is very difficult to measure accurately, because it blends in with the small angle scattering resulting from the small size of the
(13.0s) very diifaso 6.51 diffuse 6.16 4.15
3.lD
X-RAY DIFFR.\C'TIOS
STUDY O F I'ECTI?U'IC .\SD I'ECTIC ACIDS
717
DIYCUSHION
On the assumption that the chain molecules align themsrlvcs p:irallel to the direction of elongation, it, fo1lon.s that the rqii:Ltorially ticwntuatetl reflection is related to the interchain separation. This is giving a spacing of 6.5-6.9 A-i, substantiated by the fact, that this rcflection vnrips ivith thr methoxyl, alcohol, anti ivater contents, ivhcreas the meridionally accentuated rrflections are essentially invariant. From the position and lcngth of the three meridional arcs it follo\vs that those reflections must, nrisc Irom planes ivhich are either perpendicular, or w r y nearly so, to the fiber axis. T h r determination of tlir fiber axis identity period is, unfortunately, not straightforivard, hecause the position of the layer lines cannot be determined ivith certainty. Hon-rwr, it is obvious that the three meridionally accentuated reflections lie on different layer lines. I t follows, therefore, from an inspection of the values listed in taiile 3 that the fiber axis identity period cannot be less than three times the value of the 4 A. spacing. There are several reasons for suspecting that the actual value of the identity period is soniei\-hat larger than this and is in fact very nearly equal to that found for sodium pectate (15),-namely, 13.1 A. The reasons for this latter conclusion are that sodium pectate has a prominent reflection a t 4 . l i .I.ivith hexagonal indices (103) which both becomes oriented and has the same general appearance as the 4 A. spacing obtained from the prctinic acids. Of more importance, hoivever, is the fact that when sodium pectate is precipitated rapidly from solution, a product is obtained lvhich gives a diffraction pattern consisting of two prominent rings. The appearance of this photograph is very similar to that obtained from the pectinic acids. The i n t ~ n s eouter ring corresponds to a spacing of 4.17 A . , in very good agreement x i t h the value obtained from the similar ring in the pectinic acids. I t is unlikely that the identity period along a single chain in this quickly precipitated sodium pectate is any different than it is in oriented fibers; it seems more likely that, oiving to the rapid precipitation, the carboxyl groups (and therefore the sodium atoms) in adjacent chains are not arranged with sufficient regularity with respect to one another to allow the (003) reflection to appear.2 The tendency for the polygalaeturonide chains to assume a crystalline arrangement is greatest in the case of the sodium salt and least a h e n the carboxyl groups are esterified, as in high-methoxyl pectinic acids. For this reason and also because of the variable distribution in electron density, resulting from the random distribution of methoxyl groups, the (003) reflection does not appear even in fairly ne11 oriented fibers made from pectinic acids. So far w have * T h e sodium pectate sample ran readily be made to give n pattern on n-hich the (003) reflection appears by merely humidifying it. The hydrated chains are evidently able to move with respect to one another until the carboxyl groups on adjacent chains take up positions opposite one another. When a sufficient degree of crystalline regularity is attained, then, because of the threefold screw symmetry of the chains, a strong (003) reflection aDpears.
718
K. J. PALMER, R. C. MERRILL, H. S. OWENS, AND M . BALLANTYNE
not observed any reflections which can be unequivocally indexed as (001) from pectic acid fibers either. This is presumably due to the strong interaction between the polygalacturonic acid chains resulting from the presence of the free carboxyl groups, which prevents the chains from having the mobility necessary to assume a crystalline arrangement. This strong interaction between the polygalacturonic acid chains also accounts for the IOK solubility of pectic acid in water compared to the high-methoxyl pectinic acids. There is one additional piece of evidence favoring the assumption that the fiber identity period in the pectinic and pectic acids is approximately the same as in sodium pectate. When x-ray diffraction patterns were taken of a highly oriented sodium pectate fiber a t different stages of hydration, it was observed that the (003) reflection gradually disappeared and the (103) reflection (4.17 A.) became relatively much more intense. The position of the (003) reflection, however, did not vary with mater content as long as it could be observed. The photograph of completely dry sodium pectate has, like the photograph obtained from rapidly precipitated sodium pectate, the same general appearance as the photographs of pectinic acids. All of the evidence so far obtained, therefore, points to the conclusion that the fiber identity period of the polygalacturonide chain is more or less constant and has a value of about 13 A. Wuhrmann and Pilnik suggest that the fiber identity period of the polygalacturonide chain is about 8.8 A. Evidently their fibers and films were not sufficiently well oriented for them to observe the meridional reflection a t 6.1 A. which rules out this possibility.3 An identity period of 13 A. excludes the possibility of there being only two pyranose units in the identity period. The most reasonable assumption is that the polygalacturonide chain has the symmetry of a threefold screw axis. The identity period of about 13 A . is then to be expected if the pyranose rings have the trans configuration suggested for sodium pectate (14, 15) and alginic acid (1). I t is of interest in this connection that 3/2 times 8.7, the identity period for the twofold alginic acid chain, is 13.05 A., a value which is in good agreement with the value found for sodium pectate and postulated for the pectinic acids. The variation of the equatorially accentuated x-ray reflection with methoxyl content, shown in figure 2 is for samples which contain 16 per cent water, this value being taken from plots of x-ray spacings 2iersu.s moisture content. From figure 2 it is evident that the d-value calculated from the equatorial reflection is approximately a linear function of the methoxyl content and increases about 0.3 A. on going from pectic acid to a pectinic acid having a methoxyl content of 11 per cent. A methoxyl content of 11 per cent corresponds to about 76 per cent of the carboxyl groups being esterified, when, as is the case for the pectinic acids under discussion, the non-uronide content is about 18 per cent. On the a Because of the importance of this 6 1 A. meridional reflection in the deduction of the fiber identity period, it was considered desirable to obtain an x-ray photograph using strictly monochromatic radiation This was done by reflecting the x-ray beam from the cleavage face of rock salt The resulting photograph distinctly showed the presence of the 6.1 A meridional reflection
X-RAY DIFFRACTION STUDY O F PECTINIC AND PECTIC ACIDS
719
assumption that the straight-line relationship shown in figure 2 holds for complete esterification (14.5 per cent), the increase in the x-ray spacing in going from 0 to 14.5 per cent methoxyl nould be about 0.4 A. Since complete esterification 11 ould correspond to one methyl group per five atoms along the chain, this increase of 0.4 A. is in good agreement with the difference of 0.3 A. found to occur between the d-values of polybutadiene and polyisoprene (6). The number of methyl groups per chain atom in these two cases is 0 and 1/4. I t is interesting that the equatorially accentuated x-ray reflection varies in a continuous manner with the methoxyl content. This is a result of the fact that the x-ray reflection is a measure of the average interchain separation. In the case of the pectinic acids the actual interchain $stance probably varies from point to point by as much as a few tenths of an Angstrom, owing to the random distribution of methoxyl groups. This causes the equatorial reflection to be quite diffuse. The equatorial reflection from pectic acid is sharper, in agreement with the expectation that there is probably less variation in interchain separation because of the more uniform chains. The equatorial reflection is aln ays considerably more diffuse than the meridional reflection, a result which indicates that the crystalline regions are considerably longer than they are wide. This conclusion is in agreement nith the opt cal studies carried out by Van Iterson (18) and Wuhrmann and Pilnik (19) on pectin fibers. They determined the birefringence of pectin fibers immersed in media of different refractive index. From these results they were able to sho\y that the intrinsic birefringence of pectin fibers is negative with respect to the fiber axis, but that the form birefringence is positive. In other words, the crystallites are rod shaped. Rod-shaped particles have also been found to occur in pectinic acid solutions by Boehm (4), who studied their flow birefringence. The reason for the negative intrinsic birefringence of pectinic acid fibers is not known a t the present time. The negative sign cannot be due to the presence of the methoxyl groups, as has been suggested (4), because pectic acid also has a negative sign. It is unlikely that the negative sign can be due to the presence of carboxyl groups or the serpentine-like configuration of the chain, because alginic acid, which appears to have a similar chain configuration (l), has a positive sign of birefringence with respect to the fiber axis. One difference between pectinic acid and alginic acid is that the pyranose ring in the former has the a-d-configuration, while in the latter the ring has been shown to have the P-d-configuration. In pectinic acid, therefore, the C-0 bonds on the first and second carbon atoms project out on the same side of the pyranose ring, while in alginic acid, as in cellulose, these two bonds project out on opposite sides of the pyranose ring. This difference may account for the fact that the polarizability is larger in a direction perpendicular to the chain axis than parallel to the chain axis in pectinic acid, whereas the reverse is true for both alginic acid and cellulose. SUMMARY
X-ray photographs have been taken of prctinic and pectic acids in the form
720
K. J. PALMER, R. C . MERRILL, H. S. OIVENS, AND M. BALLANTYNE
of both poxvders and fibers, and their interplanar spacings have been recorded. The variation in x-ray spacings rrith methoxyl content has been determined. A fiber identity period of about 13 A. has been found for the polygalacturonide chain in all the pectinic and pectic acids so far investigated. The interchain separation increases from approximately 6.6 A . to G.94 A. when the methoxyl content varies from 0 to 11 per cent. The sign of intrinsic birefringence of pectinic and pectic acid fibers is negative. This is in contrast to fibers of alginic acid and sodium alginate, which are positive. A structural reason for the negative sign of birefringence is suggested. REFERESCES ASTBURY, R. T.: S a t u r e 155, 667 (1945). .kSTBURY, W.T., A N D BELL,F. 0 . : Tabulae Biologicae (Haag) 17,96 (1939). BAIER,W.E., ASD WILSON,C. W.: Ind. Eng. Chem. 33, 287 (1941). BOEHM,G.: Arch. euptl. Zellforsch. Gewebezucht. 22, 520 (1938-39). FRICKE, R., LOHRMANN, O., SCHRODER, W., WEITBRECHT, A , , A N D SAMMET, R . : Z. Elektrochem. 47, 374 (1941). (6) FULLER, C. S., FROSCH, C. J . , A N D PAPE,N. R.: J. Am. Chem. SOC.62, 1905 (1940). F. A,, A N D SCHNEIDER, G.: Ber. 69B, 309 (1936). (7) HENGLEIN, E. F., WAISBROT, S. W., AND RIETZ,E . : Ind. Eng. Chem. 16, 523 (1944). (8) JAIVSEN, H. VOX., AND LUNDE,G.: Kolloid-Z. 83, 202 (1938). (9) KRINGSTAD, (10) MEYER,K. H.: Satzlral and Synthetic High P o l y v w s , p. 363. Interscience Publishers, Inc., New York (1942). (11) OHN,A.: Ind. Eng. Chem. 18, 1295 (1926). (12) OWENS, H. s.,LOTZK.4R, H., MERRILL, R. C., AKD PETERSON, &f.: J. Am. Chem. SOC. (1) (2) (3) (4) (5)
Sg, 1178 (1944). (13) OWENS,H. S.,MCCREADY, R. M.,
ow.
~ H U L T Z T. , H.,
AND
MACLAY, W. D.: Ind. Eng. Chem. 36, 936
LOTZKAR, H., OWENS,H. S.,
AND
MACLAY, W. D.: J . Phys. Chem.
49, 554 (1945).
(14) PALMER, K. J., AND HARTZOG, M.B.: J. Am. Chem. Soc. 67, 1865 (1945). K. J., AND LOTZKAR, H.: J. Am. Chem. SOC. 67, 884 (1945). (15) PALMER, PALMER, K. J., AND HARTZOG, M. B.: J. Am. Chem. Soc. 67,2122 (1945). K. J., MERRILL,R. C., AND BALLANTYNE, M.: In preparation. (16) PALMER, (17) PALMER, K. J., SHAW,T. M., AND BALLANTYNE, M.: J. Polymer Sci., in press. (18) VANITERSON, G., JR.: Chem. Weekblad So, 2 (1933). K., AND PILNIK, W.: Experientia 1, 330 (1945). (19) WUHRMANN,
